Programmed cell death antagonist protein

ABSTRACT

Programmed cell death antagonist proteins and nucleic acids are provided, as well as expression vectors and host cells which contain the nucleic acids encoding the programmed cell death antagonist proteins.

FIELD OF THE INVENTION

[0001] The invention relates to proteins involved in the prevention ofprogrammed cell death, namely programmed cell death antagonist (PCDA)proteins.

BACKGROUND OF THE INVENTION

[0002] Cells become specified during development through sequentialrestriction of their potential fates. This process includes mechanismsthat monitor differentiation to eliminate, by programmed cell death,cells that have inappropriate specificity or developmental capacity, orthat are extraneous (Glucksmann, Biol Rev. 26:59-86 (1951); Saunders,Science 154:604-612 (1966)). Many aspects of tissue development rely oncell death for the selection of proper sets of cells. For example, invertebrates, massive numbers of neurons generated in early developmentbecome eliminated in late stage refinement of connections (Hamburger etal., J. Exp. Zool. 111:457-502 (1949); Oppenheim, Ann. Rev. Neurosci.14:453-501 (1991)). The mechanism is thought primarily to occur throughcompetition for trophic agents derived from target tissues, which mayreinforce appropriate patterns of innervation. Similarly, in the immunesystem, progenitor cells must generate a great diversity of cell types.The differentiation process relies heavily on regulated cell death toeliminate large numbers of cells of inappropriate reactivity (Fesus,Immunol. Lett. 30:277-282 (1991); Goldstein et al., Immunol. Rev.121:29-65 (1991)).

[0003] The mechanisms and regulation of programmed cell death have anumber of implications. For example, the regulation of programmed celldeath has implications for oncogenesis (Williams, Cell 65 1097-1098(1991)), immune disease (Ameisen et al., Immunol. Today 12:102-105(1991); Meyaard et al., Science 257:217-219 (1992)), and conditionswhere excessive cell death results in tissue damage, such as neuralinjury (Choi, Neuron 1:623-634 (1988)). Neural and immune systemdevelopment also display important programmed cell death events. Ininsects, some cell death is observed early, during neuroblastdelamination in the formation of the ventral nerve cord (Doe et al.,Dev. Biol. 111:193-205 (1985); Jimenez and Campos-Ortega, Neuron 5:81-89(1990)). In vertebrates, cell death that is not associated with targetinnervation is observed in the spinal ganglia (Hamburger et al., supra;Pannese, Neuropathol. Appl. Neurobiol. 2:247-267 (1976); Hamburger etal., J. Neurosci. 1:60-71 (1981); Carr et al., Dev. Brain Res. 2:157-162(1982)). Administration in vivo of nerve growth factor prevents nervecell death (Hamburger et al., supra), suggesting that competition forfactors may participate in the selection of neural cells even at earlydevelopmental stages. Progenitor cells of the oligodendrocyte lineagealso require certain levels of survival factors during early development(Barres et al., Cell 70:31-46 (1992)). The types of factors thatinfluence cell survival may change as the cells mature (Barres et al.,supra), suggesting that different signals may be involved in theselection of cells at different developmental stages. In the immunesystem, the elimination of cells through cell death functions atmultiple stages. T cell maturation may involve two types of selectionprocesses. Cells lacking appropriate receptors fail to be positivelyselected for further differentiation and are eliminated (Sha et al.,Nature 336:73-76 (1988); Teh et al., Nature 335:229-233 (1988)). Cellsthat do develop receptors but are self-reactive are also eliminated(Kappler et al., Nature 332:35-40 (1988); MacDonald et al., Nature332:40-45 (1988); Smith et al., Nature 337:181-184 (1989). Regulatedcell death thus appears to function together with selection to sculpt anappropriate repertoire of cells.

[0004] Programmed cell death typically occurs in conjunction withcritical differentiation events. Recent work suggests that programmedcell death is a default fate that will occur unless actively inhibited(Barres et al., supra; Raff, Nature 356:397-400 (1992). In addition,studies done in C. elegans imply that the differentiation pathway andthe cell death pathway may be uncoupled genetically (Ellis et al., Cell44:817-829 (1986); Hengartner et al., Nature 356:495-499 (1992). Genesthat function in the cell death pathway have been identified, such asthe ced genes of Caenorhabditis elegans (Ellis et al., supra; Hengartneret al., supra). However, genes are also needed to determine when duringdevelopment that pathway is activated. To coordinate differentiation anddeath, the activities of genes involved in select differentiation eventspresumably impinge on control of genes of the death pathway to repressthe suicide of appropriate cells.

[0005] The Drosophila eye is an excellent genetic system for approachingthe problem of how differentiation events and cell death interplay toachieve proper cellular development (Ready, Trends Neurosci. 12:102-110(1989); Banerjee et al., Neuron 4:177-187 (1990); Rubin, Trends Genet.7:372-377 (1991)). The adult eye is composed of some 800 repeated neuralunits called ommatidia, each containing cell types that include threephotoreceptor classes, three kinds of pigment cells, cone cells, and abristle cell complete with socket, neuron, and glial sheath. During thethird larval instar, progenitor cells commence differentiation togenerate the various cell types (Waddington et al., Proc. Roy. Soc.Lond. (B) 153:155-178 (1960)). Differentiation is marked by amorphogenetic furrow that moves from posterior to anterior across thefield of progenitor cells in the eye portion of the eye-antennalimaginal disc (Ready et al., Dev. Biol. 53:217-240 (1976)). Anterior tothe morphogenetic furrow, the progenitor cells undergo division togenerate an epithelial field for the differentiation events thatcommence with the furrow. Thus, at a given time, the disc displays atime line of development, the earliest morphologically evidentdifferentiation events being associated with the furrow. Later eventsoccur toward the posterior of the disc, where a pattern emerges ofdeveloping cell clusters. Little is known about the events before furrowformation that lead to differentiation, although cell competence,hormones, and possibly inductive interactions appear to be involved(Bodenstein, Postembryotic Development, in Insect Physiology, K. D.Roeder, ed. New York, Wiley & Sons, Inc. pp 822-865 (1953); White, J.Exp. Zool. 148:223-239 (1961); Gateff et al., Roux's Arch. Dev. Biol.176:171-189 (1975). Some cell death is a normal part of thedevelopmental process, having been observed in the eye disc duringmorphogenesis (Fristrom, Mol. Gen. Genet. 103:363-379 (1969); Spreij,Neth. J. Zool. 21:221-264 (1971); Wolff et al., Development 113:825-839(1991)).

[0006] One mutation, eya, has proven useful in the study of eyedevelopment. Flies with the eya¹ mutation show remarkable specificityfor loss of the adult compound eyes (FIGS. 1A and 1B; Sved, Dros Inf.Serv. 63:169 (1986); Renfranz et al., Dev. Biol. 136:411-429 (1989)).All other external structures appear normal, including the adult ocelli,which develop from edges of the eye imaginal discs. In the brain, thereis loss of the first optic ganglion (lamina) and reduction in size ofthe second optic ganglion (medulla), and the lobula and lobula plateshow some disorganization (FIGS. 1C and 1D). These brain defects aresimilar to those observed in other eyeless mutants and are consistentwith the influence of retinal neurons on development of the optic lobes(Power, J. Exp. Zool. 94:33-71 (1943); Meyerowitz et al., Dev. Biol.62:112-142 (1978); Fischbach, Dev. Biol. 91:1-18 (1983); Selleck et al.,Neuron 6:83-99 (1991).

[0007] In normal development, eye differentiation begins during thethird instar larval stage when the morphogenetic furrow sweeps fromposterior to anterior across the eye portion of the eye-antennal disc,leaving clusters of differentiating photoreceptor neurons in its wake(Ready et al., supra). The differentiating clusters can be visualized bystaining with monoclonal antibodies, such as neuronspecific MAb 22C10(FIG. 2E; Zipursky et al., Cell 36:15-26 (1984)). In eya¹ eye discs, thenormal expansion of the eye portion of the disc during the third instarlarval stage is arrested, and no furrow forms (FIGS. 25, 2D, and 2F).When stained with antibodies that normally highlight the differentiatingneurons, the mutant eye discs fail to show any evidence of clusterformation (FIG. 2F; also Renfranz et al., supra). In contrast, theantenna portion of the disc expands and differentiates normally. Thelarval photoreceptor organ also appears normal, and Bolwig's nerve fromthe larval visual organ (Bolwig, Vidensk. Medd. fra. Dansk. Naturh.Foren. Bd. 109:80-212 (1946) traverses the eye disc in its path into theoptic stalk, as in a normal disc. The eya¹ mutation thus appears toaffect specifically the progenitor cells that normally form the adultcompound eye.

[0008] In addition, the study of genes that function in the fly eye hasprovided insight into the developmental roles of many proteinshomologous to human genes (Greenwald et al., Cell 68:271-281 (1992).Anophthalmia, or the lack of eyes, occurs in many organisms, includinghumans (Apple et al., Ocular Pathology: Clinical applications andSelf-Assessment, St. Louis, Mosby-Year Book, Inc. (1991)). In a strikingexample, congenital aniridia in humans resembles the Small eye mutationof mouse (Glaser et al., Science 250:823-827 (1990), van der Meer-deJong et al., Genomics 7:270-275 (1990); both result from mutations ofgenes that are homologous (Hill et al, Nature 354:522-525 (1991); Ton etal., Cell 67:1059-1074 (1991); Jordan et al., Nature Genet. 1:328-332(1992)).

SUMMARY OF THE INVENTION

[0009] It is an object of the invention to provide programmed cell deathantagonist (PCDA) proteins, and variants thereof, and to produce usefulquantities of these PCDA proteins using recombinant DNA techniques.

[0010] It is a further object of the invention to provide recombinantnucleic acids encoding PCDA proteins, and expression vectors and hostcells containing the nucleic acid encoding the PCDA protein.

[0011] An additional object of the invention is to provide methods forproducing the PCDA protein, and for regulating the programmed cell deathof an organism.

[0012] In accordance with the foregoing objects, the present inventionprovides recombinant PCDA proteins, and isolated or recombinant nucleicacids which encode the PCDA proteins of the present invention. Alsoprovided are expression vectors which comprise DNA encoding a PCDAprotein operably linked to transcriptional and translational regulatoryDNA, and host cells which contain the expression vectors.

[0013] An additional aspect of the present invention provides methodsfor producing PCDA proteins which comprises culturing a host celltransformed with an expression vector and causing expression of thenucleic acid encoding the PCDA protein to produce a recombinant PCDAprotein.

[0014] Additionally provided are methods of regulating the programmedcell death of a population of cells in an organism.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 depicts the adult phenotype of eya¹ flies. Scanningelectron micrographs of normal (A) and eya¹ mutant (B) heads. Thecompound eyes are missing entirely in eya¹ flies. The three dorsalocelli and other external head structures are present. Silver-stainedhorizontal sections of a normal fly brain (C) and eya¹ mutant fly brain(D). In the mutant, the retina (r) and lamina (l) are completelymissing, the medulla (m) appears reduced in size, and the lobula (lo)and lobula plate (lp) are less organized that normal.

[0016]FIG. 2 depicts the phenotype of eya¹ in the developing eye disc.(A and B) depict eye-antennal discs from early third instar larvae,stained with neuron specific MAb22C10. The furrow is not yet present; noclusters have formed. The eye portion (e) of the eya¹ disc appearsnormal in size at this stage. Bolwig's nerve, which stains withMAb22C10, transverses the discs. (C and D) depict slightly older discsfrom early third instar larvae. The eye portion (e) of the eya¹ disc isstill expanding. (E) depicts the wild-type-eye-antennal disc from a late(crawling) third instar larva stained with MAB22C10. The morphogenicfurrow (f) is halfway across the eye portion (e) of the disc. Thedeveloping neuronal clusters posterior to the furrow are highlighted bythe antibody staining. (F) depicts the eya¹ mutant eye-antennal discfrom a late (crawling) third instar larva stained with MAb22C10. Theantennal portion (a) of the disc has expanded normally, but the eyeportion (e) of the disc is much smaller than normal. No furrow isformed; neural clusters fail to develop, illustrated by the lack ofstaining with MAb22C10. Bolwig's nerve is present. Bar=50 μm.

[0017]FIG. 3 depicts compound eye phenotypes of flies expressing variousalleles of eya. (A-D) are scanning electron micrographs. (E-H) aretangential sections, anterior to the right. (A) and (E) are wild typeflies.

[0018] The regular ommatidial pattern is shown by the arrow in (E). Therhabdomeres of seven photoreceptor cells are visible: six outer, andinner seventh. The rhabdomere of the eighth photoreceptor is beneaththat of the seventh. The photoreceptor cells are surrounded by apigmented lattice. (B) and (F) are mild allele eya^(E3) heterozygouswith eya¹. The eye is slightly reduced and rough. Photoreceptor cellsare occasionally missing (arrow in F) and the pigmented lattice is lessregular than in wild type. Many ommatidia appear normal (white arrow in(F)). (C) and (G) are intermediate allele eya^(E4) heterozygous witheya¹. The eye is reduced and rough. Many ommatidia contain the fullcomplement of photoreceptor cells (white arrow in (G)), although cellsare sometimes missing (black arrow) and the pigmented lattice is lessorganized than in wild type. (D) and (H) are severe allele eya^(E1)heterozygous with eya¹. In flies expressing this allelic combination,the eye is greatly reduced and rough (D), or missing altogether.Although the ommatidial pattern of such severely reduced eyes showsdisorganization (H), many ommatidia contain all the photoreceptor celltypes (white arrows) and the pigmented lattice is present.

[0019]FIG. 4 depicts the eye disc phenotypes of larvae expressingvarious alleles of eya. Eye portions of eye-antennal discs from crawlingthird instar larvae. (A-D) depict MAB 22C10 staining, which highlightsneural clusters. (E-H) depicts acridine orange staining, whichhighlights dead cells. Arrows mark the morphogenic furrow. (A and E) arewild type. (A) posterior to the furrow clusters are developing. (E) deadcells appear as bright dots of fluorescence. A small amount of celldeath occurs in a band just anterior to the furrow. (B and F) are mildallele eya^(E3) heterozygous with eya¹. (B) Many neural clusters form,consistent with the only slightly reduced eye in the adult (see FIG.3B). (F) Anterior to the furrow (arrow), an increase in the amount ofcell death occurs, highlighted by acridine orange staining. Theincreased cell death occurs in the same region of the disc just anteriorto the furrow where normally some cell death occurs. (C and G) areintermediate allele eya^(E4) heterozygous with eya¹. (C) Fewer clustersform. Anterior to the furrow, condensed and refractile dying cells arevisible. (G) Acridine orange highlights the increased degeneration thatoccurs in a broad band anterior to the furrow (arrows). (D and H) aresevere allele eya^(E1) heterozygous with eya¹. (D) Many fewer clustersform in the eye disc. Anterior to the furrow, condensed dead cells arevisible. Inset: higher magnification view of condensed and refractilebodies anterior to the furrow. Arrows highlight a few examples. (H)Acridine orange staining highlights the great increase in cell deaththat occurs anterior to the furrow in the disc. Bars are 50 μm.

[0020]FIG. 5 depicts the cell death in an eye disc of an eya mutant thatforms a reduced eye. (A) eye portion of an eye-antennal disc from athird instar larva expressing an intermediate allele eya^(E9)heterozygous with eya¹. Anterior to the furrow (arrow) cell death isseen in the basal region of the disc (boxed area). Dead cells fragmentinto electron-dense bodies; these appear to become engulfed bysurrounding cells. Posterior to the furrow, where the clusters aredifferentiating, no cell death is seen. (B) Higher magnification viewfrom the box in (A). Within dead cell fragments, intact cellularorganelles may be seen (arrow points toward what appears to be anucleus). Also visible are fragments of dead cells within other cells.Bar in (A)=10 μm, bar in (B)=2 μm. Anterior to the right.

[0021]FIG. 6 depicts the molecular analysis of the eya chromosomalregion. (A) shows the molecular organization of the eya region. Therestriction map from overlapping cosmid and phage clones shows Notl (N),BamH1 (B), EcoR1 (E), Sal1 (S), and Xbal (X) sites. This region residesin cytological region 26E on chromosome 2. Above the restriction map areindicated the DNA restriction fragments within which the breakpoints ofsix X-ray-induced alleles fall. Also shown is the location of thebreakpoint of an X-ray allele of dpp, T(2;3)DTD46, that has onebreakpoint in 26E and that fails to complement eya mutations.Illustrated below the restriction map are the intron-exon structures ofprototypes of the two classes of cDNA that span the region. The start(ATG) and stop (TGA) codons correspond to those of the longest potentialopen reading frames. The initiation sites of transcription have not beendetermined. (B) Northern blot analysis. Northern blot of poly(A)+RNAisolated from wild-type embryos (0-24 hours), adult bodies and heads,and adult heads of the eya¹ mutant. Each lane has 7.5 μg of RNA. Theblot was probed with the Not1-EcoR1 restriction fragment indicated by anasterisk on the restriction map in (A). A transcription unit of 3.5 kbis detectable in embryos and adult heads; its intensity is reduced inthe heads of the eya¹ mutant. (C) is a Northern blot of eye discpoly(A)+RNA, isolated from 200 mid-third instar larval eye-antennaldiscs from wild type and the eya¹ mutant, probed with the entire type 1cDNA. A transcript of 3.5 kb is detectable in the wild type, but ismissing in the mutant.

[0022]FIG. 7 depicts the nucleic acid and protein sequence of the eyagene. (A) is the nucleic acid sequence of the type 1 cDNA, with thestart and stop of the coding region shown and the corresponding aminoacid sequence (B) is N-terminus of the type II cDNA, and thecorresponding amino acid sequence. For the type I cDNA, the sequence atthe proposed start site has a 2/4 bp match with the Drosophilatranslation initional consensus (Cavener, Nucl. Acids. Res. 15:1353-1361(1987). For the type II cDNA, the match is 3/4 bp. The first 19 aminoacids of the type I cDNA and the first 25 of the type II are generatedby alternative splicing. Within the amino acid sequence common to bothcDNA classes, three charged clusters (Brendel et al., Proc. Natl. Acad.Sci. USA 89:2002-2006, 1992) are arranged as a basic stretch (solidunderline), an acidic stretch (double underline), then a second basicstretch (solid underline). Five regions (amino acids 83-203, 236-256,409-429, 489-509, and 671-691) are predicted to be hydrophobic β-helicalregions by the algorithm of Eisenberg et al., J. Mol. Biol. 179:125-142(1984). The opa repeat spans amino acids 40-62. A possible PEST proteindegradation sequence (Rogers et al., Science 234:364-368 (1986);Rechsteiner, Sem. Cell Biol. 1:433-440 (1990)) is underlined with adashed line, and potential cyclic nucleotide-dependent, protein kinase Cand tyrosine kinase phosphorylation sites are circled. Amino acids 18-23are a candidate for a nuclear localization signal (Chelsky et al., Mol.Cell. Biol. 9:2487-2492 (1989)). The 3′ untranslated region isapproximately 500 nt and has two AATAAA sites (boxed) that could serveas polyadenylation signal sequences. Within the 3′ tail region are sitATTTA repeats (underlined), which are found in the 3′ untranslatedregions of dynamically expressed genes and are implicated in rapidmessage turnover (Shaw et al., Cell 46:659-667 (1986)). The intron sites(carets) were determined by comparing the genomic sequence with that ofthe cDNAs.

[0023]FIG. 8 depicts the protein and transcript expression of the eyagene in eye discs. (A-E) are eye discs stained with a mouse polyclonalantiserum directed against the eya protein. (F-G) are transcriptexpression using digoxigenin labeling of the type I cDNA. (A and B) showthat protein expression begins during the second instar, remains onprior to furrow formation in third instar, and appears graded withexpression stronger in the posterior region of the disc than theanterior. Two eye discs are visible in (A). (C and D) As the furrowforms and progresses across the disc, expression remains strong in theregion just anterior to the furrow (arrows). The protein is expressedmore weakly to the anterior of the eye portion of the disc. Posterior tothe furrow, expression continues. (E) Longitudinal section of a latethird instar eye disc stained with the polyclonal antiserum, detectedwith a secondary antibody conjugated to horseradish peroxidase. Onset ofexpression is anterior to the furrow (arrow). The protein is localizedto nuclei. (F) Longitudinal section of a late third instar eye disclabeled by in situ hybridization. Dark-field image superimposed on abright-field image. Onset of transcript signal (white dots) is in theregion anterior to the furrow (arrow), and continues posterior to thefurrow, wherein it is expressed primarily in the basal region. (G) Insitu expression in a whole-mount preparation of an eye disc from a wildtype crawling third instar larva. One flap of the disc is curled over atthe bottom (outlined with dashes). Transcript expression begins anteriorto the furrow (arrow), and is weaker in the posterior region of thedisc. Bar (A-D and G)=50 μm. Bar in (E) and (F) is 20 μm.

[0024]FIG. 9 depicts the restoration of development of the eye in eyamutants through expression of the hsp-eya minigene by heat shock. (A-C)are scanning electron micrographs. (D-E) are tangential sections of theeye. (A and D) are the wild-type eye and ommatidial pattern. (B) eya²mutant harboring an hsp-eya minigene insert, raised without heat shock.The mutant is completely eyeless. (C and E) eya² mutant bearing anhsp-eya minigene insert, heat pulsed during the larval stages ofdevelopment. Both the external eye morphology and the internal ommatidiaare restored. Anterior to the right.

DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention provides novel PCDA proteins. As usedherein, a “PCDA protein” is a protein which exhibits a repressive orantagonistic effect on programmed cell death. That is, a population ofcells which otherwise would undergo programmed cell death are preventedfrom dying by the presence of the PCDA protein. It is to be understoodthat the precise mechanism of the regulatory action of the PCDA proteinis not known, although three possible mechanisms are proposed. The PCDAprotein may repress cell death, thus allowing cells to enter thedifferentiation pathway; the PCDA protein may promote differentiation,which represses cell death; or the PCDA protein both promotesdifferentiation and represses cell death.

[0026] The PCDA proteins and the nucleic acid encoding the PCDA proteinsof the present invention are homologous to the Drosophila amino acid andnucleic acid sequence shown in FIG. 7, as outlined below, and it is thishomology which serves as the major distinguishing characteristic of aPCDA protein. In addition, a PCDA protein may have one or more of thefollowing characteristics. First, the PCDA proteins are localized in thenucleus. In addition, the expression of PCDA protein at certain times inthe development and differentiation pathway will prevent the programmedcell death of a population of cells. Alternatively, the repression orinhibition of the PCDA protein may expedite the programmed cell death ofa cell population.

[0027] Accordingly, the PCDA proteins of the present invention find usein a number of applications. For example, PCDA proteins are useful asreagents in diagnostic assays for the presence of PCDA protein orantibodies to PCDA protein, or when insolubilized in accord with knownmethods as agents for the purification of PCDA protein antibodies fromantisera or hybridoma culture. PCDA proteins are also useful as animmunogen or hapten to produce antibodies to PCDA protein. Theseantibodies are useful in the diagnostic assay of PCDA protein andelucidation of the localization of the PCDA protein within a tissue ororganism.

[0028] At the broadest level, the PCDA proteins of the present inventionare useful as regulators or modulators of programmed cell death. Thusthe expression of the nucleic acid encoding PCDA protein, or theaddition of the PCDA protein directly, prevents programmed cell death ina cell population. Conversely, the inhibition of a PCDA protein isuseful in cell populations in which cell death is desirable, for examplein tumor cells. Accordingly, inhibitors such as antisense nucleic acidsencoding PCDA protein of the present invention are useful.

[0029] The nucleic acids of the present invention which encode PCDAproteins are also useful in the generation of transgenic animals, usedas models for the elucidation of programmed cell death function andeffect. For example, transgenic animals which do not contain afunctional PCDA protein gene are useful in the determination of the roleof programmed cell death in development. These transgenic animals arealso useful as cancer models, since many types of cancer exhibit a lossof cellular programmed cell death, with a corresponding increase incancerous cellular growth and proliferation. Transgenic animals withincreased production of the PCDA protein are also useful.

[0030] The nucleic acids of the present invention are also useful ingene therapy, wherein nucleic acid is targeted to a cell type or tissueto prevent cell death. This includes both the addition of a PCDA proteingene or the deletion of a PCDA protein gene.

[0031] In accordance with the above applications, the PCDA proteins andnucleic acids are defined and generated as outlined below in detail.

[0032] The present invention provides novel PCDA proteins. A PCDAprotein nucleic acid or PCDA protein is initially identified bysubstantial nucleic acid and/or amino acid sequence homology to thesequences shown in Figures X and Y. Such homology can be based upon theoverall nucleic acid or amino acid sequence.

[0033] In the case of the protein, the overall homology of the proteinsequence to the amino acid sequence shown in FIG. 7 is preferablygreater than about 40%, more preferably greater than about 60% and mostpreferably greater than 80%. In some embodiments the homology will be ashigh as about 90 to 95 or 98%. This homology will be determined usingstandard techniques known in the art, such as the Best Fit sequenceprogram described by Devereux et al., Nucl. Acid Res. 12:387-395 (1984).The alignment may include the introduction of gaps in the sequences tobe aligned. In addition, for sequences which contain either more orfewer amino acids than the protein shown in FIG. 7, it is understoodthat the percentage of homology will be determined based on the numberof homologous amino acids in relation to the total number of aminoacids. Thus, for example, homology of sequences shorter than that shownin FIG. 7 will be determined using the number of amino acids in theshorter sequence.

[0034] In the case of the nucleic acid, the overall homology of thenucleic acid sequence is commensurate with amino acid homology but takesinto account the degeneracy in the genetic code and codon bias ofdifferent organisms. Accordingly, the nucleic acid sequence homology maybe either lower or higher than that of the protein sequence. Thus thehomology of the nucleic acid sequence as compared to the nucleic acidsequence of FIG. 7 is preferably greater than 40%, more preferablygreater than about 60% and most preferably greater than 80%. In someembodiments the homology will be as high as about 90 to 95 or 98%.

[0035] cDNA analysis of the PCDA protein from Drosophila reveals that atleast two separate cDNAs exist, the putative result of alternatesplicing. These cDNAs, type I and type II, differ in the use of 5′ exons(FIG. 6A). Accordingly, PCDA proteins may exhibit N-terminalheterogeneity and truncation.

[0036] The PCDA proteins and nucleic acids of the present invention arerecombinant. As used herein, “nucleic acid” may refer to either DNA orRNA, or molecules which contain both deoxy- and ribonucleotides. Thenucleic acids include genomic DNA, cDNA and oligonucleotides includingsense and anti-sense nucleic acids. Specifically included within thedefinition of nucleic acid are anti-sense nucleic acids. An anti-sensenucleic acid will hybridize to the nucleic acid sequence shown in FIG.7, but may contain ribonucleotides as well as deoxyribonucleotides.Generally, anti-sense nucleic acids function to prevent expression ofmRNA, such that a PCDA protein is not made. The nucleic acid may haveintrons which are not transcribed, in the case of genomic DNA, forexample. The nucleic acid may be double stranded, single stranded, orcontain portions of both double stranded or single stranded sequence. Bythe term “recombinant nucleic acid” herein is meant nucleic acid,originally formed in vitro by the manipulation of nucleic acid byendonucleases, in a form not normally found in nature. Thus an isolatedPCDA protein gene, in a linear form, or an expression vector formed invitro by ligating DNA molecules that are not normally joined, are bothconsidered recombinant for the purposes of this invention. It isunderstood that once a recombinant nucleic acid is made and reintroducedinto a host cell or organism, it will replicate non-recombinantly, i.e.using the in vivo cellular machinery of the host cell rather than invitro manipulations; however, such nucleic acids, once producedrecombinantly, although subsequently replicated non-recombinantly, arestill considered recombinant for the purposes of the invention.

[0037] Similarly, a “recombinant protein” is a protein made usingrecombinant techniques, i.e. through the expression of a recombinantnucleic acid as depicted above. A recombinant protein is distinguishedfrom naturally occurring protein by at least one or morecharacteristics. For example, the protein may be isolated away from someor all of the proteins and compounds with which it is normallyassociated in its wild type host. The definition includes the productionof a PCDA protein from one organism in a different organism or hostcell. Alternatively, the protein may be made at a significantly higherconcentration than is normally seen, through the use of a induciblepromoter or high expression promoter, such that increased levels of theprotein is made. Additionally, the protein may be made at a differentstage in development than normal or as a result of new experimentalconditions, through the use of a temperature sensitive or heat shockpromoter. Alternatively, the protein may be in a form not normally foundin nature, as in the addition of an epitope tag or amino acidsubstitutions, insertions and deletions.

[0038] Also included with the definition of PCDA protein are PCDAproteins from other organisms, which are cloned and expressed asoutlined below.

[0039] A PCDA protein nucleic acid from an organism other thanDrosophila can be readily identified by standard methods utilizing allor part of the sequence shown in FIG. 7. For example, labelled probescorresponding to the sequence of FIG. 7 can be used for hybridization todetect the presence of an PCDA protein gene in a particular organism. Inaddition, such probes can be used to screen genomic or cDNA libraries orto identify one or more bands containing all or part of the PCDA proteingene by hybridization to an electrophoretically separated preparation ofgenomic DNA digested with one or more restriction endonucleases.

[0040] The length of the probes will vary depending upon the organismscreened and the amount of expected homology. Generally probes may be atleast about 10 bases in length, and will generally be no more than about50 bases in length, with a preferred range of about 15 to 30 bases.However, it is to be understood that much longer probes may be used,comprising all or a significant part of the sequence shown in FIG. 7,for example to clone the Drosophila protein.

[0041] The hybridization conditions will vary depending upon the probeused, and will be ascertainable by one skilled in the art using routineexperimentation. Generally, low stringency conditions, e.g. 4-5×SSC at50° C., will be used in the initial screening of genomes other than thatof Drosophila. High stringency conditions may also be used, i.e. 0.1×SSCat 65° C., when greater homology is expected. Accordingly, nucleic acidswhich hybridize, under either low or high stringency conditions, to thesequence shown in FIG. 7 are within the scope of the invention.

[0042] In the case of anti-sense nucleic acids, an anti-sense nucleicacid is defined as one which will hybridize to all or part of thesequence shown in FIG. 7. Generally, the hybridization conditions usedfor the determination of anti-sense hybridization will be highstringency conditions, such as 0.1×SSC at 65° C.

[0043] Once the PCDA protein nucleic acid is identified, it can becloned and, if necessary, its constituent parts recombined to form theentire PCDA protein nucleic acid. Once isolated from its natural source,e.g., contained within a plasmid or other vector or excised therefrom asa linear nucleic acid segment, the recombinant PCDA protein nucleic acidcan be further used as a probe to identify and isolate other PCDAprotein nucleic acids. It can also be used as a “precursor” nucleic acidto make modified or variant PCDA protein nucleic acids and proteins.

[0044] Using the nucleic acids of the present invention which encodePCDA protein, a variety of expression vectors are made. The expressionvectors may be either self-replicating extrachromosomal vectors orvectors which integrate into a host genome. Generally, these expressionvectors include transcriptional and translational regulatory nucleicacid operably linked to the nucleic acid encoding the PCDA protein.“Operably linked” in this context means that the transcriptional andtranslational regulatory DNA is positioned relative to the codingsequence of the PCDA protein in such a manner that transcription isinitiated. Generally, this will mean that the promoter andtranscriptional initiation or start sequences are positioned 5′ to thePCDA protein coding region. The transcriptional and translationalregulatory nucleic acid will generally be appropriate to the host cellused to express the PCDA protein; for example, transcriptional andtranslational regulatory nucleic acid sequences from Drosophila will beused to express the PCDA protein in Drosophila. Numerous types ofappropriate expression vectors, and suitable regulatory sequences areknown in the art for a variety of host cells.

[0045] In general, the transcriptional and translational regulatorysequences may include, but are not limited to, promoter sequences,leader or signal sequences, ribosomal binding sites, transcriptionalstart and stop sequences, translational start and stop sequences, andenhancer or activator sequences. In a preferred embodiment, theregulatory sequences include a promoter and transcriptional start andstop sequences.

[0046] Promoter sequences encode either constitutive or induciblepromoters. The promoters may be either naturally occurring promoters orhybrid promoters. Hybrid promoters, which combine elements of more thanone promoter, are also known in the art, and are useful in the presentinvention.

[0047] In addition, the expression vector may comprise additionalelements. For example, the expression vector may have two replicationsystems, thus allowing it to be maintained in two organisms, for examplein mammalian or insect cells for expression and in a procaryotic hostfor cloning and amplification. Furthermore, for integrating expressionvectors, the expression vector contains at least one sequence homologousto the host cell genome, and preferably two homologous sequences whichflank the expression construct. The integrating vector may be directedto a specific locus in the host cell by selecting the appropriatehomologous sequence for inclusion in the vector. Constructs forintegrating vectors are well known in the art.

[0048] In addition, in a preferred embodiment, the expression vectorcontains a selectable marker gene to allow the selection of transformedhost cells. Selection genes are well known in the art and will vary withthe host cell used.

[0049] The PCDA proteins of the present invention are produced byculturing a host cell transformed with an expression vector containingnucleic acid encoding a PCDA protein, under the appropriate conditionsto induce or cause expression of the PCDA protein. The conditionsappropriate for PCDA protein expression will vary with the choice of theexpression vector and the host cell, and will be easily ascertained byone skilled in the art through routine experimentation. For example, theuse of constitutive promoters in the expression vector will requireoptimizing the growth and proliferation of the host cell, while the useof an inducible promoter requires the appropriate growth conditions forinduction. In addition, in some embodiments, the timing of the harvestis important. For example, the baculoviral systems used in insect cellexpression are lytic viruses, and thus harvest time selection can becrucial for product yield.

[0050] A recombinant PCDA protein may be expressed intracellularly orsecreted. The PCDA protein may also be made as a fusion protein, usingtechniques well known in the art.

[0051] In a preferred embodiment, the PCDA protein is purified orisolated after expression. The PCDA proteins may be isolated or purifiedin a variety of ways known to those skilled in the art depending on whatother components are present in the sample. Standard purificationmethods include electrophoretic, molecular, immunological andchromatographic techniques, including ion exchange, hydrophobic,affinity, and reverse-phase HPLC chromatography, and chromatofocusing.For example, the PCDA protein may be purified using a standard antibodycolumn. Ultrafiltration and diafiltration techniques, in conjunctionwith protein concentration, are also useful. For general guidance insuitable purification techniques, see Scopes, R., Protein Purification,Springer-Verlag, NY (1982). The degree of purification necessary willvary depending on the use of the PCDA protein. In some instances nopurification will be necessary.

[0052] Appropriate host cells include yeast, bacteria, archebacteria,fungi, and insect and animal cells, including mammalian cells. Ofparticular interest are Drosophila melangaster cells, Saccharomycescerevisiae and other yeasts, E. coli, Bacillus subtilis, SF9 cells, C129cells, 293 cells, Neurospora, BHK, CHO, COS, and HeLa cells,immortalized mammalian myeloid and lymphoid cell lines.

[0053] In a preferred embodiment, PCDA proteins are produced in insectcells. Expression vectors for the transformation of insect cells, and inparticular, baculovirus-based expression vectors, are well known in theart. Briefly, baculovirus is a very large DNA virus which produces itscoat protein at very high levels. Due to the size of the baculoviralgenome, exogenous genes must be placed in the viral genome byrecombination. Accordingly, the components of the expression systeminclude: a transfer vector, usually a bacterial plasmid, which containsboth a fragment of the baculovirus genome, and a convenient restrictionsite for insertion of the PCDA protein; a wild type baculovirus with asequence homologous to the baculovirus-specific fragment in the transfervector (this allows for the homologous recombination of the heterologousgene into the baculovirus genome); and appropriate insect host cells andgrowth media.

[0054] After inserting the nucleic acid encoding the PCDA protein intothe transfer vector, the vector and the wild type viral genome aretransfected, usually through co-transfection, into an insect host cellwhere the vector and viral genome are allowed to recombine. Methods forintroducing heterologous DNA into the desired site in the baculovirusare known in the art. For example, a preferred embodiment uses aninsertion into the polyhedrin gene, such that recombination results in aloss of the polyhedrin protein. Then the insertion proceeds viahomologous double recombination. Homologous recombination generallyoccurs at low frequency (between 1% and about 5%). Thus, the majority ofthe virus produced after cotransfection is wild-type. However, due tothe very high levels of polyhedrin production by the native virus, thewild-type accumulated polyhedrin protein forms highly refractileocclusion bodies that are readily visualized under a light microscope.Therefore, cells infected with recombinant viruses which insert into thepolyhedrin gene lack occlusion bodies, making visualization ofrecombinants with a light microscope possible.

[0055] Materials and methods for baculovirus/insect cell expressionsystems are commercially available in kit form; for example the “MaxBac”kit from Invitrogen in San Diego.

[0056] Baculovirus transfer vectors usually contain a baculoviruspromoter. A baculovirus promoter is any DNA sequence capable of bindinga baculovirus RNA polymerase and initiating the downstream (5′ to 3′)transcription of a coding sequence, i.e. the PCDA protein gene, intomRNA. Of particular use as promoters are the promoters from the genesencoding the viral polyhedrin protein and the p10 protein.

[0057] A signal sequence may also be included in the insect cellexpression vector. These signal sequences can be derived from secretedinsect or baculovirus proteins, such as the polyhedrin gene.Alternatively, since the signals for mammalian cell post-translationalmodifications (such as signal peptide cleavage, proteolytic cleavage,and phosphorylation) appear to be recognized by insect cells, and thesignals required for secretion and nuclear accumulation also appear tobe conserved between the invertebrate cells and vertebrate cells,leaders of non-insect origin may be used. Mammalian expression systemsare also known in the art and are used in one embodiment. A mammalianpromoter is any DNA sequence capable of binding mammalian RNA polymeraseand initiating the downstream (3′) transcription of a coding sequencefor PCDA protein into mRNA. A promoter will have a transcriptioninitiating region, which is usually place proximal to the 5′ end of thecoding sequence, and a TATA box, using a located 25-30 base pairsupstream of the transcription initiation site. The TATA box is thoughtto direct RNA polymerase II to begin RNA synthesis at the correct site.A mammalian promoter will also contain an upstream promoter element,typically located within 100 to 200 base pairs upstream of the TATA box.An upstream promoter element determines the rate at which transcriptionis initiated and can act in either orientation. Of particular use asmammalian promoters are the promoters from mammalian viral genes, sincethe viral genes are often highly expressed and have a broad host range.Examples include the SV40 early promoter, mouse mammary tumor virus LTRpromoter, adenovirus major late promoter, and herpes simplex viruspromoter.

[0058] Typically, transcription termination and polyadenylationsequences recognized by mammalian cells are regulatory regions located3′ to the translation stop codon and thus, together with the promoterelements, flank the coding sequence. The 3′ terminus of the mature mRNAis formed by site-specific post-translational cleavage andpolyadenylation. Examples of transcription terminator and polyadenlytionsignals include those derived form SV40.

[0059] Some genes may be expressed more efficiently when introns arepresent. Several cDNAs, however, have been efficiently expressed fromvectors that lack splicing signals. Thus, in some embodiments, thenucleic acid encoding the PCDA protein includes introns.

[0060] The methods of introducing exogenous nucleic acid into mammalianhosts, as well as other hosts, is well known in the art, and will varywith the host cell used. Techniques include dextran-mediatedtransfection, calcium phosphate precipitation, polybrene mediatedtransfection, protoplast fusion, electroporation, encapsulation of thepolynucleotide (s) in liposomes, and direct microinjection of the DNAinto nuclei.

[0061] In a preferred embodiment, PCDA protein is produced in yeastcells. Yeast expression systems are well known in the art, and includeexpression vectors for Saccharomyces cerevisiae, Candida albicans and C.maltosa, Hansenula polymorpha, Kluyveromyces fragilis and K. lactis,Pichia guillerimondii and P. pastoris, Schizosaccharomyces pombe, andYarrowia lipolytica. Preferred promoter sequences for expression inyeast include the inducible GAL1,10 promoter, the promoters from alcoholdehydrogenase, enolase, glucokinase, glucose-6-phosphate isomerase,glyceraldehyde-3-phosphate-dehydrogenase, hexokinase,phosphofructokinase, 3-phosphoglycerate mutase, pyruvate kinase, and theacid phosphatase gene. Yeast selectable markers include ADE2, HIS4,LEU2, TRP1, and ALG7, which confers resistance to tunicamycin; the G418resistance gene, which confers resistance to G418; and the CUP1 gene,which allows yeast to grow in the presence of copper ions.

[0062] Once expressed and purified if necessary, the PCDA proteins, aswell as the PCDA protein nucleic acids as outlined below, may beadministered to an animal or organism to regulate programmed cell deathin a cell population. By “regulating programmed cell death” or“modulating programmed cell death” or grammatical equivalents herein ismeant the alteration of a pattern of programmed cell death. Generally,this regulation will be the repression or prevention of programmed celldeath, such that a cell population slated to undergo programmed celldeath is prevented from doing so, resulting in the retention of a viablecell population. The regulation of programmed cell death does notrequire that the complete population are prevented from dying, but thatsome subset of the population is prevented.

[0063] By “cell population” or “a population of cells” or grammaticalequivalents herein is meant a collection of cells, generally relateddevelopmentally or as cells of a tissue. For example, progenitor cellsof a certain type may be considered a cell population, or eye tissuecells. In a preferred embodiment, the cell population which is preventedfrom undergoing programmed cell death is the progenitor eye cells.

[0064] The administration of the PCDA protein is done in a variety ofways. Generally, the PCDA proteins of the present invention can beformulated according to known methods to prepare pharmaceutically usefulcompositions, whereby the PCDA protein is combined in admixture with apharmaceutically acceptable carrier vehicle. Suitable vehicles and theirformulation are well known in the art. Such compositions will contain aneffective amount of the PCDA protein together with a suitable amount ofvehicle in order to prepare pharmaceutically acceptable compositions foreffective administration to the host. The composition may include salts,buffers, carrier proteins such as serum albumin, targeting molecules tolocalize the PCDA protein at the appropriate site or tissue within theorganism, and other molecules.

[0065] In a preferred embodiment, the PCDA protein is administered invivo to a developing organism to prevent anophthalmia or aniridia. Thisadministration may be accomplished either through the administration ofthe PCDA protein itself, or through the administration, by gene therapy,of nucleic acid encoding the PCDA protein, resulting in the in vivoexpression of the protein. For example, the expression of the PCDAprotein gene after microinjection into eyeless Drosophila mutant embryosallows the formation of eyes, due to the prevention of the programmedcell death of the eye progenitor cells.

[0066] Also contemplated within the scope of the invention is the use ofinhibitors or repressors of the PCDA protein to increase programmed celldeath. For example, the use of an inhibitor of PCDA protein in certaintumor cells can allow the repression of cell death to be avoided, thusresulting in the elimination of the tumor through cell death. Thisinhibitor may be an anti-sense nucleic acid, for example, or anantibody. The anti-sense nucleic acid will be all or part of the nucleicacid complement of the coding nucleic acid shown in FIG. 7. The creationand administration of anti-sense nucleic acids is known in the art.

[0067] In a preferred embodiment, as noted above, expression of the PCDAprotein occurs as a result of gene therapy, that is, the administrationof nucleic acid encoding a PCDA protein to an organism. Thus, the PCDAprotein will be produced endogenously in the organism, rather thanadministered in a protein form. The gene therapy may be done at anembryonic stage of the organism, such that the germ cells of theorganism contain the PCDA protein nucleic acid, resulting in atransgenic organism, or at a later stage of development to specificsomatic cells, such that only a particular tissue or portion of a tissuecontains the PCDA protein nucleic acid. Techniques for gene therapy arewell known in the art, as are the techniques for the creation oftransgenic organisms. By the term “transgenic organism” herein is meanttransgenic animals, such as mammals, as well as insects.

[0068] It is to be understood that the administration of a PCDA proteinnucleic acid in gene therapy may take several forms, all of which areincluded in the scope of the present invention. The nucleic acidencoding a PCDA protein may be administered in such a manner as to addthe PCDA protein nucleic acid to the genome of the cell or the organism.For example, administering a nucleic acid encoding a PCDA protein, underthe control of a promoter which results in an increase over backgroundexpression of PCDA protein, results in the incorporation of the nucleicacid into the genome of the cell or the organism, such that increasedlevels of PCDA protein are made. For example, this may be done to a cellpopulation which is slated to undergo an undesirable level of programmedcell death, to preserve the cells. Alternatively, an anti-sense nucleicacid encoding a PCDA protein, that is, a nucleic acid which willhybridize to all or part of the coding strand for the PCDA protein, maybe administered to decrease the amount of PCDA protein expressed in acell population. Optionally, a nucleic acid encoding a PCDA protein maybe used to delete the gene from the cell or organism, resulting in adecrease in the amount of PCDA protein made by the cell. This may bedone, for example, to a population of cancer cells to derepressprogrammed cell death. Techniques for the creation of deletion mutantsis well known in the art.

[0069] Construction of appropriate expression vehicles and vectors forgene therapy applications will depend on the organism to be treated andthe purpose of the gene therapy. The selection of appropriate promotersand other regulatory DNA will proceed according to known principles,based on a variety of known gene therapy techniques. For example,retroviral mediated gene transfer is a very effective method for genetherapy, as systems utilizing packaging defective viruses allow theproduction of recombinants which are infectious only once, thus avoidingthe introduction of wild-type virus into an organism. Alternativemethodologies for gene therapy include non-viral transfer methods, suchas calcium phosphate co-precipitation, mechanical techniques, forexample microinjection, membrane fusion-mediated transfer via liposomes,as well as direct DNA uptake and receptor-mediated DNA transfer.

[0070] Also included within the definition of PCDA proteins of thepresent invention are amino acid sequence variants. These variants fallinto one or more of three classes: substitutional, insertional ordeletional variants. These variants ordinarily are prepared by sitespecific mutagenesis of nucleotides in the DNA encoding the PCDAprotein, using cassette mutagenesis or other techniques well known inthe art, to produce DNA encoding the variant, and thereafter expressingthe DNA in recombinant cell culture as outlined above. However, variantPCDA protein fragments having up to about 100-150 residues may beprepared by in vitro synthesis using established techniques. Amino acidsequence variants are characterized by the predetermined nature of thevariation, a feature that sets them apart from naturally occurringallelic or interspecies variation of the PCDA protein amino acidsequence. The variants typically exhibit the same qualitative biologicalactivity as the naturally occurring analogue, although variants can alsobe selected which have modified characteristics as will be more fullyoutlined below.

[0071] While the site or region for introducing an amino acid sequencevariation is predetermined, the mutation per se need not bepredetermined. For example, in order to optimize the performance of amutation at a given site, random mutagenesis may be conducted at thetarget codon or region and the expressed PCDA protein variants screenedfor the optimal combination of desired activity. Techniques for makingsubstitution mutations at predetermined sites in DNA having a knownsequence are well known, for example, M13 primer mutagenesis. Screeningof the mutants is done using assays of PCDA protein activities, as isknown by those in the art. For example, nucleic acid encoding thevariants may be put under the control of a heat shock promoter andinjected into embryos, and heat pulse experiments done to evaluate theeffect of the variant PCDA protein on the development of an organism.Alternatively, the variant PCDA protein may be expressed and itsbiological characteristics evaluated, for example its binding to DNA.

[0072] Amino acid substitutions are typically of single residues;insertions usually will be on the order of from about 1 to 20 aminoacids, although considerably larger insertions may be tolerated.Deletions range from about 1 to 30 residues, although in some casesdeletions may be much larger.

[0073] Substitutions, deletions, insertions or any combination thereofmay be used to arrive at a final derivative. Generally these changes aredone on a few amino acids to minimize the alteration of the molecule.However, larger changes may be tolerated in certain circumstances.

[0074] When small alterations in the characteristics of the PCDA proteinare desired, substitutions are generally made in accordance with thefollowing chart: Chart I Exemplary Original Residue Substitutions AlaSer Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro HisAsn, Gln Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile PheMet, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

[0075] Substantial changes in function or immunological identity aremade by selecting substitutions that are less conservative than thoseshown in Table I. For example, substitutions may be made which moresignificantly affect: the structure of the polypeptide backbone in thearea of the alteration, for example the alpha-helical or beta-sheetstructure; the charge or hydrophobicity of the molecule at the targetsite; or the bulk of the side chain. The substitutions which in generalare expected to produce the greatest changes in the polypeptide'sproperties are those in which (a) a hydrophobic residue, e.g. seryl orthreonyl, is substituted for (or by) a hydrophobic residue, e.g. leucyl,isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline issubstituted for (or by) any other residue; (c) a residue having anelectropositive side chain, e.g. lysyl, arginyl, or histidyl, issubstituted for (or by) an electronegative residue, e.g. glutamyl oraspartyl; or (d) a residue having a bulky side chain, e.g.phenylalanine, is substituted for (or by) one not having a side chain,e.g. glycine.

[0076] The variants typically exhibit the same qualitative biologicalactivity as the naturally-occurring analogue, although variants also areselected to modify the characteristics of the polypeptide as needed. Forexample, epitope coding sequences may be added or protease recognitionsites eliminated, without altering the biological activity of thepolypeptide. The evaluation of the new characteristics of thepolypeptide will vary with the alteration. For example, the addition ofan epitope coding sequence may be evaluated by antibody binding studies,and the deletion of a protease recognition site evaluated by treatmentwith protease followed by gel electrophoresis or a biological activityassay. Other changes may be evaluated using assays for PCDA proteinactivities and characteristics, as will be known to those skilled in theart of PCDA proteins. These guidelines allow one skilled in the art tosuccessfully evaluate the effect and characteristics of any insertion,deletion or substitution of amino acid sequence in a PCDA protein.

[0077] Alternatively, the variant may be designed such that thebiological activity of the PCDA protein is altered. To this end, thereare several regions within the protein which may be subjected to aminoacid substitutions, deletions or insertions. For example, the PCDAprotein from Drosophila exhibits three charged clusters (Brendel, supra)arranged as a basic stretch at amino acids 449 to 471 (unless otherwisenoted, all amino acids are numbered according to the type I numbering ofFIG. 7, an acidic stretch at amino acids 528 to 563, and a basic stretchat amino acids 600 to 629. Multiple charge clusters such as these areuncommon and are generally associated with regulatory proteins,including protooncogenes, transcription factors, and various types ofreceptors (Brendel et al., Proc. Natl. Acad. Sci. USA 86:5698-5702(1989), Karlin et al., Oncogene 5:85-95 (1990)). In addition, the PCDAprotein gene from Drosophila contains 5 regions which are predicted tobe hydrophobic α-helical regions using the algorithm of Eisenberg, etal., supra. These regions are found at amino acids 83-103, 236-256,409-429, 489-509, and 671-691. An opa repeat spans amino acids 40-62. Aputative PEST protein degradation sequence (Rogers, et al., supra,Rechsteiner, supra) is found at amino acids 373-388. Putative cyclicnucleotide dependent protein kinase C and tyrosine kinasephosphorylation sites are found at Thr447, Thr453, Thr456, Ser615,Ser658, Tyr706, and Thr718. Finally, amino acids 18-23 are a candidatefor a nuclear localization signal (Chelsky et al., supra). It isinteresting to note that these amino acids span the beginning of thecommon sequence region. Thus amino acid variants within all theseregions are contemplated.

[0078] Important functional domains within the protein may also beidentified by comparing the amino acid sequence of variants, generatedby mutagenesis, with the wild-type gene.

[0079] Variants are assayed using the techniques outlined in Example 4,as are known to those skilled in the art. For example, injection ofembryos with nucleic acid encoding the variants can result in theintegration of the nucleic acid encoding the variant, with a resultingalteration in the phenotype of the organism.

[0080] Also included with the definition of PCDA protein for thepurposes of the present invention are fragments of PCDA protein ortruncated PCDA protein. For example, C- or N-terminal truncations, orboth, may be made which retain full or partial biological activity. Forexample, the alternate splicing seen in the Drosophila melangaster eyesuggests that N-terminal truncations are tolerated.

[0081] Alternatively, N- or C-terminal truncations, or both, maydecrease or eliminate the biological activity of the enzyme. However,variant PCDA proteins which display low or negligible biologicalactivity are included in the definition of PCDA protein if the variantexhibits at least 40% homology with full-length PCDA protein and sharesat least one immunological epitope in common with the full-length PCDAprotein.

[0082] PCDA protein derivatives that are not biologically active butwhich are capable of cross-reacting with antisera or antibodies tobiologically active PCDA protein, as well as biologically active PCDAproteins, are useful in several applications. These derivatives areuseful as reagents in diagnostic assays for PCDA protein or antibodiesto PCDA protein, or when insolubilized in accord with known methods asagents for the purification of PCDA protein antibodies from antisera orhybridoma culture supernatants. These derivatives are also useful asimmunogens to biologically active PCDA protein. In addition, thesederivatives are useful in assays of PCDA protein functions, such asassays of binding interactions, for example interactions between a PCDAprotein and DNA sequences, and in assays to determine the role of PCDAproteins in cellular functioning.

[0083] The following examples serve to more fully describe the manner ofusing the above-described invention, as well as to set forth the bestmodes contemplated for carrying out various aspects of the invention. Itis understood that these examples in no way serve to limit the truescope of this invention, but rather are presented for illustrativepurposes.

EXAMPLES Example 1 Allelic Analysis of the eya Gene

[0084] Fly Strains

[0085] Flies were cultured on standard cornmeal medium at 25° C. Mutantstrains are listed in Lindsley et al., The Genome of Drosophilamelanogaster, San Diego: Academic Press (1992). The wild-type strainnormally used was Canton-S. Additional wild-type strains used to examinecell death were Urbana-S, Lausanne-S, Oregon-R, and Oregon-R-C, kindlyprovided by E. B. Lewis (California Institute of Technology).

[0086] Alleles of the eya Gene

[0087] Differentiation occurs in a timed manner in normal eye discs,progressing from posterior to anterior across the discs with the advanceof the morphogenetic furrow. Thus, for eye mutants, knowing when celldeath occurs relative to the furrow would allow the determination of thedevelopmental stage at which the cells die. Owing to the extremephenotype of the eya¹ mutant, in which no furrow occurs, it is notpossible to determine this stage. Therefore new eye alleles with lessextreme eye phenotypes to place the cell death relative to the criticalevents in eye differentiation were generated. Additional alleles of theeye gene were isolated by screening for mutations that failed tocomplement the eye phenotype of eya¹, using techniques outlined below.Several mutants contributed by other laboratories were also determinedto be eye alleles by genetic mapping and failure of complementation.Most of the newly generated alleles are lethal or semilethal whenhomozygous. The lethality is embryonic (N. M. B., unpublished data) andfails to complement an independently isolated embryonic lethal mutation,clift (Nusslein-Volhard et al., Roux's Arch. Dev. Biol. 193:267-282(1984). Flies bearing the clift allele in trans to the viable eya¹allele show a severe eye phenotype. In addition, some eya alleles andinterallelic combinations show reduced or absent ocelli, abnormalmorphology of the adult brain, and female sterility. These resultssuggest that the eya gene has functions beyond its role in eyedevelopment; the eye function appears, in part, independently mutable.The gene also displays interallelic complementation.

[0088] Four eya alleles generated in other laboratories were obtained.These are eya¹ (Sved, supra), eya² (also eya^(ph) or eya^(pinhead); TMcQuirre, Rutgers University), eya³ (D. Mohler, University of Iowa, IowaCity), and eya⁴ (ey-2 of Eisenberg et al., Dros. Inf. Serv. 70:266-268(1991). Once the eya gene was mapped cytologically, other mutations inthe region for complementation were tested.

[0089] This analysis revealed that T(2; 3)DTD46 has a breakpoint in theeya gene (Gelbart, Proc. Natl. Acad. Sci. USA 79:2636-2640 (1982) andthat the mutation clift is allelic (Nüsslein-Volhard et al., supra).

[0090] 35 new alleles were generated using ethyl methanesulfonate,X-rays, and P element hybrid dysgenesis as mutagens, as outlined below.All were isolated by failure to complement the eye phenotype of the eya¹mutation. Putative alleles were recovered over a second-chromosomebalancer chromosome.

[0091] The eye phenotypes of the new alleles, in trans to the viableeya¹ mutation, comprise a phenotypic series (FIG. 3). The mutants can beclassified as: mild, in which the eyes are rough, but only slightlysmaller than normal size (FIGS. 3B and 3F); intermediate, in which botheyes always form, but are rough and reduced in size (FIGS. 3C and 3G);severe, in which the eyes are rough, much reduced, and frequently absentfrom one or both sides of the head (FIGS. 3D and 3H); and completely andconsistently eyeless, like the eya¹ mutant (see FIG. 10). In tangentialsections of reduced and rough eyes of heteroallelic combinations, someommatidia lack the full complement of photoreceptor cells (FIGS. 3E-3H).However, no obvious specific subset of cells is missing as alleleseverity increases, unlike in a mutant like sevenless (Harris et al., J.Physiol. 256:415-439 (1976). Even in severely reduced eyes, fullcomplements of photoreceptor cells frequently form within individualommatidia (FIG. 3H). These results suggest that eye mutations reduce thenumber of progenitor cells available for recruitment into the developingeye, rather than specifically eliminating any particular cell type(s).

[0092] Mutagenesis was as follows. Approximately 2050 spd⁹ male flieswere mutagenized with ethyl methanesulfonate (Lewis et al, Dros. Inf.Serv. 43:193-194 (1968) and mated to eya¹ virgin females, and theprogeny were raised at 29° C. Of 90,000 flies screened, 12 independentalleles, eya^(E1) to eya^(E12), were isolated, one of which displayedtemperature sensitivity. For the X-ray screen, about 1000 spd males weresubjected to 4500 rads, then mated to eya¹ virgin females. From 57,000total progeny screened, 16 alleles were isolated. Eight of these havecytologically visible rearrangements: eya^(X1) with In(2L)26E;36-37,eya^(X3) with T(2;3)26E;86C, eya^(X8) with In(2L)26E;39, eya^(X10) withT(2;3)26E;67A, eya^(X5) with T(2;3)26E;70A, eya¹⁶with T(2;3)26E;3L;heterochromatin, and ey^(X9) and eya^(X11) with complex breakpoints thatwere not determined.

[0093] Seven alleles (eya^(P1) to eya^(P7)) were isolated in twoindependent screens using P element hybrid dysgenesis, after Robertsonet al., Genetics 118:461-470 (1988). In the first screen, an Spchromosome was used as the parental chromosome; in the second screen,the alleles were generated on an isogeneic wild-type second chromosomeor on the Birmingham second chromosome. In both screens, progeny fromthe Birmingham 2 by P[ry⁺Δ2-3](99B) (Laski et al., Cell 44:7-19 (1986)cross were mated to virgin eya¹ females and screened for failure ofcomplementation. In the first screen, one allele (eya^(P1)) was isolatedof 200,000 F2 flies scored. In the second screen, 70,000 F2 flies werescored, and six additional alleles recovered. None of these alleles wasassociated with a P element at the chromosomal location of eya, analyzedby chromosomal in situ hybridization.

[0094] The eye disc phenotypes of eya alleles were studied in trans tothe eya¹ chromosome marked with Cy. Cy was recombined onto the eya¹chromosome by R. Hackett from the CyO second chromosome balancer. Thiswas possible since the eya¹ mutation arose on an inversion (Sved, supra)with breakpoints on 2 L similar to those of the balancer. Thischromosome is referred to as Cy,eya¹.

Example 2 Isolation of Genomic and cDNA Clones

[0095] Of 16 X-ray-induced alleles, 6 with cytologically visiblerearrangements in the polytene chromosomes of the larval salivary glandshad a common breakpoint on the left arm of chromosome 2, in cytologicalregion 26E. This location is consistent with the position of the eyegene determined by meiotic recombination, which placed both the eyephenotype (mapped for all cytologically normal alleles) and thelethality (mapped for eya^(E4) and eya^(P1)) between dp and spd on theleft arm of the second chromosome. The region was cloned in a 60 kbgenomic walk, as described below. Restriction fragments were testedagainst eya breakpoint alleles by in situ hybridization to the salivarychromosomes and by Southern blot analysis, as described below. Thechromosomal breaks of the eya mutants all fell within a 25 kb region(FIG. 6A).

[0096] Standard molecular techniques were from Sambrook et al.,Molecular Cloning, in A Laboratory Manual (Cold Spring Harbor, N.Y.:Cold Spring Harbor Laboratory Press) (1989). Flies mutant for variouseya alleles were mated to Canton-S, and polytene chromosomes of thelarval salivary glands were dissected, stained with orcein, and analyzedfor visible cytological rearrangements. A DNA probe for the 26Ecytological region was obtained from Drosophila yeast artificialchromosome DY81 7, which spans into the region (kindly provided by I.Duncan, Washington University at St. Louis; Garza et al., Science246:641-646 (1989). DNA was isolated from a 5 ml culture of DY817. Thiswas digested with EcoRV and Hincll, ligated, and amplified with primersto the yeast artificial chromosome vector, to generate by inversepolymerase chain reaction Drosophila DNA probes for the ends of theyeast artificial chromosome, as described by Ochman et al.,Amplification of flanking sequences by inverse PCR, in PCR Protocols: AGuide to Methods and Applications, Innis, et al., eds., San Diego:Academic Press, pp 219-227 (1990). Primers used were5′-GCGATGCTGTCGGAATGGAC-3′ and 5′-GTTGGTTTAAGGCGCAAGACT-3′ for the EcoRVside, and 5′-CGAGTCGAACGCCCGATCTC-3′ and 5′-AGGAGTCGCATAAG-GGAGAG-3′ forthe Hincll side. The polymerase chain reactions were run on a 1.5% lowmelt agarose gel, and the major bands were isolated. These were labeledby nick translation with bio-16-dUTP (ENZO Biochemicals) and used toprobe Canton-S larval salivary gland chromosome squashes to determinethe cytological site of hybridization. The signal was detected by thestreptavidin-peroxidase complex (Detek-1-HRP Kit from ENZOBiochemicals). A 740 bp probe that hybridized in cytological region 26Ewas labeled by random primer reaction and used to screen a cosmidlibrary from an isogeneic strain (kindly provided by J. Tamkun,University of California, Santa Cruz) and a Drosophila genomic phagelibrary (Stratagene). Overlapping clones of the walk were oriented bychromosomal in situ analysis. The locations of the breakpoint fragmentsin the alleles were determined by DNA Southern analysis and chromosomalin situ analysis. cDNAs were identified and isolated from Drosophilahead cDNA libraries (Itoh et al., Proc. Natl. Acad. Sci. USA83:4081-4085 (1986); Zinsmaier et al., J. Neurogenet. 7:15-29 (1990)).cDNAs and genomic fragments in the region were subcloned into thepBluescript vector (Stratagene) and transformed or electroporated intoXL-1 blue cells (Stratagene). Sequencing of cDNA and genomic clones wasperformed on cesium chloride-banded DNA preparations or minipreparationsof plasmid DNA, using 5-deaza-dGTP (US Biochemicals) and makingsequential primers. Sequence comparison with the EMBL and GenBank databases was performed according to Pearson et al., Proc. Natl. Acad. Sci.USA 85:2444-2448 (1988) and Altschul et al., J. Mol. Biol. 215:403-410(1990). Sequence analysis was performed using the GCG Sequence AnalysisSoftware Package (Devereux et al., supra), the SAPS (statisticalanalysis of protein sequences) program (Brendel et al., supra), thePEST-FIND program (Rogers et al., supra; Rechsteiner, Adv. Enzyme Reg.27:135-151 (1988)), and the algorithm of Eisenberg et al., J. Mol .Biol. 179:125-142 (1984) for potential a-helical transmembrane regions.

[0097] Two types of cDNAs were obtained from the screening of the cDNAlibraries from poly(A)+RNA purified from adult heads. The cDNAscorrespond to alternatively spliced products identical at the 3′ end,but differing in the use of 5′ exons (FIG. 6A). Both cDNA classesrecognize transcripts of 3.5 kb on Northern blots of poly(A)+RNA fromwild-type adult heads. Transcripts of 3.5 kb were detected on Northernblots of poly(A)+RNA isolated from third instar larval eye discpreparations using the cDNAs as probes (FIG. 6C). These transcripts werereduced in intensity in eye discs from eya¹ larvae. To determine whetheradditional alleles demonstrated transcripts of altered size, the cDNAswere used to probe poly(A)+RNA prepared from heads of eya mutants. Of 12alleles that exhibit no cytologically visible chromosomalrearrangements, 1 allele showed an altered transcript of 4.7 kb(eya^(X2)). These results show that the transcripts are products of theeya gene.

[0098] Sequence comparison of the type I and II cDNAs showed that the5′-most sequences differ, while the ˜2800 bp of 3′ sequence are shared.Conceptual translation of the type I cDNA, starting from the firstpotential initiation codon, revealed a single large open reading frameof 2280 nt (FIG. 7). The predicted protein has 760 residues, with apredicted molecular mass of 80 kd. The open reading frame of thealternatively spliced type II cDNA is 2298 bp. As a result of thealternative splicing, the proteins predicted for the two classes of cDNAdiffer in their extreme amino-terminal sequences (FIG. 7).

[0099] Comparison with proteins in the GenBank and EMBL data basesrevealed that the eya protein is novel. Study of the amino acid sequencesuggests that the protein may be divided into two domains. The aminoterminal half, corresponding roughly to amino acids 1-436 (numbers inreference to the type I cDNA class), is rich in alanine, glycine, andserine, and shows several single amino acid repeats, including apolyglutamine-rich stretch corresponding to an opa repeat (Wharton etal., Cell 40:55-62 (1985)). The carboxy-terminal half of the protein hasfewer amino acid repeats and contains three charged stretches that arearranged as basic- acidic-basic domains, although the protein as a wholeis predicted to be relatively neutral (predicted pl=6.8). In addition tothese features, the protein is predicted to have five hydrophobica-helical stretches (FIG. 7 legend; Eisenberg et al., supra). Given thenuclear localization of the gene product (below), these stretches areunlikely to represent transmembrane domains.

Example 3 Analysis of RNA and In Situ Expression

[0100] Nucleic acid was extracted using a modification of the procedureof Sargent et al., Dev. Biol. 114:238-246 (1986). Adult flies andembryos were collected and stored frozen at −70° C. Eye-antennal disccomplexes, with mouth hooks attached, were dissected and immediatelyfrozen on dry ice. Eye disc poly (A) RNA was prepared using theMicro-FastTrack System (Invitrogen Corporation). For adult tissue, bodyparts ground to powder in liquid nitrogen were mixed with 4.2 Mguanidine isothiocyanate, 0.5% Sarkosyl, 25 mM Tris (pH 8.0), 0.7%2-mercaptoethanol, at a ratio of 1 g of tissue per 30 ml. Samples wereDounce homogenized. Tubes were prepared with 1 vol of phenol extractionbuffer (100 mM Tris [pH 8.01], 10 mM EDTA, 1% SDS) layered over 2 vol ofphenolchloroform. One volume of tissue suspension was added, mixed, andcentrifuged. The suspension was extracted twice more with an equalvolume of phenol-chloroform, then once with chloroform. The nucleic acidwas precipitated with an equal volume of isopropanol.

[0101] Poly(A)⁺ RNA was isolated over oligo(dT) columns by the FastTrackmRNA isolation protocol (Invitrogen Corporation). The RNA was thenseparated on 1% agarose-formaldehyde gels and blotted onto reinforcednitrocellulose paper (Schleicher & Schuell) Probes were made by randomprimer labeling or by making single-stranded RNA probes using the T7 andT3 promoters of the pBluescript vector. The direction of transcriptiondetermined by single-strand probes agreed with the structure of thecDNAs by sequence analysis.

[0102] The results show that a transcript of 3.5 kb that is disrupted byat least five of the breakpoint alleles; it is present in wild-typeadult heads and is less abundant in heads of eya¹ flies (FIG. 6B). It isalso present in embryos.

[0103] To study the temporal and spatial expression patterns oftranscription of the gene in normal eye discs, whole-mount tissue insitu hybridization was done as follows, using a modification of theprotocol of Tautz et al., Chromosoma 98:81-85 (1989). Probes of bothtype I and type II cDNAs were made by random primer labeling, usingdigoxigenin-11-dUTP (Boehringer Mannheim). After hybridization anddetection of the signal (Boehringer Mannheim Genius Kit), discs weremounted in Aquamount (Lerner Laboratories). Some discs were postfixed 30min in 1% glutaraldehyde, 1% paraformaldehyde in 0.1 M phosphate buffer(pH 7.4), dehydrated, and embedded in Epon. Serial sections of 0.8 μmwere cut, lightly stained with toluidine blue, and mounted underPermount for photography.

[0104] The results show that strong eya RNA expression occurs in theregion of the disc just anterior to the furrow (FIGS. 8F and 8G). Thetranscript is also present posterior to the furrow, primarily in thebasal region of the disc. In addition, two sites of expression arepresent on the edge of the eye disc, far anterior to the furrow, nearthe antennal disc (FIG. 8G), that probably correspond to the progenitorsof the ocelli, which are derived from this area (Bryant, Patternformation in imaginal discs, in The Genetics and Biology of Drosophila,Ashburner et al., eds. London: Academic Press, pp 229-335, (1978). Theocelli are, in fact, missing in some heteroallelic combinations of eyamutants (unpublished data).

[0105] To determine the protein expression pattern, mouse polyclonalantiserum against a fusion protein was raised as follows. A 2.3 kb Smalfragment of type I cDNA was subcloned into the Smal site of expressionvector pGEX-2 (Smith et al., Gene 67:31-40 (1988). Sequence analysisconfirmed that the insert was in frame. The fusion protein was of thepredicted size of 86 kd, of which 60 kd corresponded to thecarboxy-terminal 551 amino acids of the cDNA and 26 kd to glutathioneS-transferase. The protein sequence produced is common to both types Iand II cDNAs. A 500 ml culture was grown 1.5 hr at a 1:10 dilution in LBplus ampicillin, then the fusion protein was induced by addition of 1 mMisopropyl β-D-thiogalactopyranoside. The cells were collected bycentrifugation at 4,000×g for 5 min, then resuspended in 7.5 ml of 50 mMTris(pH 8.0), 1 mM EDTA, 100 mM sodium chloride, 1 mMphenylmethylsulfonyl fluoride. Lysozyme was added to 0.3 mg/ml, and thesample incubated at room temperature for 20 min. DNAase I was then addedto 3 mg/ml for another 30 min. The sample was spun at 5,000×g for 10min, and resuspended in 9 ml of 50 mM Tris(pH 8.0), 10 mM EDTA, 100 mMsodium chloride, 1 mM phenylmethylsulfonyl fluoride, 1% Triton X-100,and allowed to sit 10 min on ice. This was spun at 10,000×g for 15 min,resuspended in 4 ml of Laemmli sample buffer, and boiled. One milliliterof the sample was run on a preparative 0.1% SDS-7.5% polyacrylamide gel,stained 10 min in 0.05% Coomassie blue in water, destained, and theregion of the gel with the fusion protein sliced out. Mice wereimmunized ten times with 50 μg of fusion protein per injection, and tailsera were collected. The antigen recognized by the polyclonal antiserumis expressed ectopically in heat-shocked transformant larvae carryingthe hsp-eya minigene construct.

[0106] The pattern of antiserum staining revealed that the eya proteinfirst becomes detectable in cells of the eye portion of the eye-antennaldisc during the second larval instar; the expression is graded, beingstronger in cells in the posterior and edges of the eye portion of thedisc, than in cells in the anterior and central region (FIG. 8A). Thisstaining pattern persists to the third larval instar (FIG. 80). As themorphogenetic furrow forms, the protein stays on in a graded manneranterior to it, with strongest expression just anterior (FIGS. 8C and8D). Protein expression persists in the cells as the furrow passes(FIGS. 8D and 8E). Posterior to the furrow, the expression is patterned,reflecting the array of developing neural clusters. Cells with nuclei inthe basal region of the epithelium, which are presumably cells not yetrecruited into developing neural clusters, show expression of theprotein; in the apical region of the disc, expression is strong in somecells of the differentiating neural clusters (FIGS. 8D and 8E). Theprotein seems to be localized to the nucleus; it is not present in thenucleolus (FIG. 8E). In animals homozygous for the eya¹ allele, whichhave normal ocelli but are eyeless (see FIG. 10), eya protein expressionoccurs in the eye discs only in the ocellar progenitors (data notshown). The lack of detectable protein expression in the eye progenitorcells of the mutant indicates that it is null or a severe hypomorph foreya gene activity in these cells. The onset of expression of bothtranscript and protein in the progenitor cells anterior to themorphogenetic furrow, where the increase in cell death occurs in eyamutants, is consistent with critical functioning of the gene in eventsthat precede furrow formation.

[0107] We also examined the expression pattern of the eya gene elsewherein normal animals (unpublished data). This analysis revealed a specificand dynamic expression pattern in the embryo, beginning with the onsetof zygotic gene expression in the cellular blastoderm, and continuing inregions of the developing head and in segments. The gene does not appearto be expressed in the embryonic eye primordia or in eye discs duringthe first larval instar. In addition, the eya gene is expressed inselect other tissues in patterns that may be related to the embryoniclethal and adult phenotypes of select eya alleles. Thus, far from beingubiquitously expressed, the eya gene shows select expression in specificregions of the developing and adult organism.

Example 3 Histology and Immunocytochemical Analysis

[0108] The apparent arrest in development of the eye disc epithelium ineya¹ animals could result from a block in cell division preventing thegeneration of progenitor cells. Alternatively, the cells might begenerated but could fail to differentiate normally. To distinguishbetween these possibilities, mutant and wild-type larvae were labeled invivo with pulses of 5-bromodeoxyuridine (BrdU). BrdU is incorporatedinto the DNA of dividing cells in S phase; these cells can then bevisualized by immunofluorescence using antibodies specific for BrdU(Truman et al., Dev. Biol. 125:145-157 (1988). In normal eye discs,dividing cells were labeled in a scattered pattern in the regionanterior to the furrow where progenitor cells are generated. At thefurrow, DNA synthesis was absent. Posterior to the furrow, cell divisionresumed in a restricted band, reflecting pattern formation events (Readyet al., supra). In eya¹ mutant eye discs at the early third instarlarval stage, the amount of cell division was similar to that in theregion anterior to the furrow in normal eye discs of the same stage(data not shown). This result suggests that cells in the eya¹ eye discdo divide; a lack of cell division to generate progenitor cells seemsnot to be the primary defect.

[0109] Given that progenitor cells divide in the eya¹ eye disc, thepossibility that a lack of normal differentiation results from the lossof cells by death was examined. In normal third instar larval eye discsrelatively little cell death is present (see FIG. 4E). In contrast, eyediscs of eya¹ animals reveal a dramatic increase in cell death duringthe third instar larval stage. Cells are present in the eya¹ eye discthat appear condensed and refractile by light microscopy, reminiscent ofcells dying by programmed cell death in C. elegans (see FIG. 4D; Sulstonet al., Dev. Biol. 56:110-156 (1977)). Dead cellular material alsofluoresces brightly when stained with acridine orange (Spreij, supra).Such staining reveals a great increase in the number of dead cells ineye discs of the eya¹ mutant (see FIG. 4H). In the electron microscope,eya¹ mutant eye discs show electron dense condensed fragments of cellsand many examples of these fragments engulfed within other cells (seeFigures). These morphological features are characteristic of cells dyingby programmed cell death (Wyllie et al., Int. Rev. Cytol. 68:251-306(1980); Kerr et al., Apoptosis, in Perspectives on Mammalian Cell Death,Pollen, ed. (Oxford: Oxford University Press), pp 93-128 (1987); Clarke,Anal. Embryol. 181:195-213 1990). These results suggest that the defectin the eya¹ mutant is a loss by cell death of eye progenitor cells.

[0110] Scanning electron microscopy was performed on unfixed flies andon flies stored in 70% ethanol, dehydrated to 100% ethanol, and criticalpoint dried. In both cases, flies were coated with gold-palladium 80:20.For tangential eye sections, fly heads were fixed in 1% glutaralde-hyde,1% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), dehydrated, andembedded in Epon (Polysciences, Inc.). Sections of 0.7 μm thickness werecut, lightly stained with 1% toluidine blue, 1% borax solution in water,and mounted in Permount (Fisher Scientific). To examine eye discs bytransmission electron microscopy, discs were dissected in 0.1 Mphosphate buffer (pH 7.4), fixed for 1 hr in 1% glutaraldehyde, 1%paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Tissue was stained1 hr in 1% osmium, 0.5% uranyl acetate, dehydrated through ethanol, andembedded in Epon, and serial 0.08 μm sections were cut.

[0111] Silver staining of adult heads was performed according to theprocedure of Meyerowitz et al., supra, as modified by Harte et al.,Genetics 101:447-501 (1982) and K. Stark (Yale University), who kindlyshared many helpful suggestions.

[0112] Labeling and detection of cells in S phase in wild-type and eya¹mutant eye discs was performed as described by Truman et al., supra,with the following modifications. Staged larvae were labeled in vivo for2 hr with BrdU at 1 mg/ml. Primary anti-BrdU antibody (Becton-Dickinson)incubation was overnight at 4° C. with slow shaking, and the secondaryantibody was fluorescein conjugated (Cappel Laboratories).

[0113] Eye discs were stained with acridine orange according to theprotocol of Spreij, supra). Dead cellular material fluoresces brightlywhen stained with acridine orange. After dissection in Ringer'ssolution, discs were incubated 5 min in 1.6×10⁻⁶ M acridine orange inRinger's solution, rinsed, and viewed with fluorescence optics.

[0114] Eye discs were dissected, fixed, and stained with MAb 22C10overnight, as described by Van Vactor et al., Cell 67:1145-1155 (1991),with some modifications. The horseradish peroxidase-conjugated secondaryantibody (Bio-Rad Laboratories) was used at a 1:50 dilution, andsubstrate was detected with 0.5 mg/ml diaminobenzidine plus 2 mg/mlNiCl. For visualization of the eya protein with the polyclonal antiseraon whole-mount preparations of eye discs, a modification of the protocolof Renfranz et al., supra, was used. Eye discs were dissected intris-buffered saline (100 mM Tris [pH 7.5], 130 mM NaCl, 5 mM KCl, 5 mMNaN₃, 1 mM EGTA), fixed 30 min in 2% paraformaldehyde in tris-bufferedsaline, then permeabilized 30 min in 0.5% Nonidet P-40 in tris-bufferedsaline. After rinsing in tris-buffered saline, discs were incubated30-60 min in tris-buffered saline plus 5% normal goat serum (VectorLaboratories). Primary antibody staining was at 1:500-1:1000 intris-buffered saline plus 5% normal goat serum for 60 min, discs wererinsed 30 min in tris-buffered saline plus 5% normal goat serum, thenincubated in fluorescein-conjugated secondary antibody at 1:500 (Cappel)diluted in tris-buffered saline plus 5% normal goat serum. After washing30 min in tris-buffered saline plus 5% normal goat serum, discs weremounted in 90% glycerol plus 0.1% phenylene, diamine, and viewed byfluorescence microscopy. Localization by light microscopy of the eyaprotein with the mouse polyclonal antiserum was by the protocol abovefor MAb 22C10, with some modifications. The antiserum was used at a1:500 dilution. Following substrate detection, discs were postfixed 20min in 1% paraformaldehyde plus 1% glutaraldehyde in 0.1 M phosphatebuffer (pH 7.4), and embedded in Epon. Longitudinal sections of 0.7 μmthickness were cut, and tissue was lightly stained with 1% toluidineblue, 1% borax solution in water, prior to mounting in Permount.

[0115] eya mutants with reduced adult eyes exhibit neuraldifferentiation in the eye disc, as illustrated by staining with MAb22C10 (FIGS. 4A-4D). However, consistent with reduced eyes in the adult,fewer neural clusters form in the eye mutant larval eye disc. Withincreasing severity of the phenotype, the number of clusters that formdecreases. In eye discs of larvae expressing intermediate and severeallele combinations, where very reduced numbers of ommatidia form, theclusters develop in the posterior-most region of the eye disc (FIGS. 4Cand 4D). When examined with BrdU labeling, eye discs of larvaeexpressing intermediate allele combinations show cell division bothanterior to the furrow and in a band posterior to the furrow, reflectingaspects of pattern formation seen in normal eye discs (data not shown).In eyeless allelic combinations, no furrow is seen, no clustersdifferentiate, and dramatic increases in cell death occur in the eyediscs, as in the eya¹ mutant. Staining for dead cellular materialrevealed a great increase in the number of dead cells in eye discs ofthe eya¹ mutant (see FIG. 4H). In the electron microscope, eya¹ mutanteye discs show electron dense condensed fragments of cells and manyexamples of these fragments engulfed within other cells (see FIG. 5).These morphological features are characteristic of cells dying byprogrammed cell death, rather than necrosis, since necrotic cells swell,losing membrane activity. (Wyllie et al., supra; Kerr et al., supra;Clarke, supra.) In contrast, dying cells in eya mutant discs appear tocondense, becoming refractile by light microscopy. By transmissionelectron microscopy, condensed bodies containing intact cellularorganelles are seen; some bodies are engulfed within healthy cells, asis typical of programmed cell death in other systems where the debris israpidly removed by phagocytosis.

[0116] Cell death in the mutant discs overlaps a stage during which someprogenitor cell death normally occurs prior to furrow formation in eyemorphogenesis (see FIG. 4E; Spreij, supra; Wolff et al., supra). Thissuggests that loss of eya activity skews the distribution of cells intoa normally occurring cell death pathway. In eye discs of larvae bearingmild to severe alleles, only a fraction of the progenitor cellsundergoes cell death; the remaining cells appear to proceed normally toform a furrow with clusters differentiating behind it. The incidence ofcell death anterior to the furrow appears to correlate inversely withthe final number of ommatidia. Since mutation of the eya gene does notseem to affect division of the progenitor cells, the data are consistentwith lack of eya activity resulting in a switch in cell fate from thepathway of differentiation to that of cell death. Moreover, theincreased cell death in the mutant discs appears highly restricted tothe region anterior to the furrow: dying cells are not observed withinthe furrow, and no increase in cell death is seen posterior to it. Theseobservations suggest the existence of a regulated mechanism, actingprior to furrow formation, that allows some cells rather than others toundergo pattern formation events. The data suggest that a selectionpoint may normally occur prior to furrow formation when some cells,presumably inappropriate or extraneous, are eliminated; the eya geneappears to function critically in this selection process.

[0117] In addition, the result that the eye disc of the eya¹ mutantclosely resembles that of the eya¹ allele in trans to lethal alleles, isconsistent with the eya¹ mutant being a severe hypomorph or null for anecessary eye function of the eye gene.

[0118] Since the eye discs of mutants showing partial eye developmentdisplay a quasi-normal framework of differentiation, the cell deathrelative to the morphogenetic furrow can be placed. Examination of eyediscs of such eye mutant combinations revealed dramatic increases incell death restricted to the region anterior to the furrow (FIGS. 4E-4H;FIG. 5). This could be observed by differential interference contrastoptics, in which dead cells appear condensed and refractile (FIGS. 4Cand 4D), and highlighted by fluorescence microscopy with acridine orange(FIGS. 4E-4H). In several different wild-type strains of D. melanogasterexamined, we consistently find some degree of cell death just anteriorto the furrow, varying from a small amount to a thin band, as in FIG. 4E(also Spreij, supra; Wolff et al., supra). In eye discs from eya alleliccombinations that form mildly reduced eyes, the increase in cell deathalso occurs as a band just anterior to the furrow, in the same regionwhere the low level of cell death normally takes place (compare FIG. 4Ewith 4F). This suggests that loss of eye function may shunt cells into anormally occurring cell death pathway anterior to the furrow. In eyediscs from intermediate and severe allele combinations, which form moreseverely reduced eyes, the amount of cell death is greater, covering abroader region anterior to the furrow (FIGS. 4G and 4H). Normal discsalso show some cell death in the differentiating region of the discposterior to the furrow during the third instar (Spreij, supra; Wolff etal., supra); this cell death is not increased by eya mutations (FIGS.4E-4H).

[0119] The ultrastructure of the dying cells in the region anterior tothe furrow was examined by transmission electron microscopy. In bothnormal (data not shown) and mutant discs (FIG. 5), the morphologicalchanges appear characteristic of programmed cell death (Wyllie et al.,supra; Kerr et al., supra; Clarke, supra.) Dead cells condense intoelectron-dense bodies containing well-preserved cellular organelles.These bodies seem to be engulfed by surrounding cells, so that thedebris is cleared by the time the furrow passes. Consistent withobservations using acridine orange, the elevated level of cell death isrestricted to the region of the disc anterior to the furrow (FIG. 5A).In eye discs of larvae bearing mild allele combinations, the cell deathis rapid: since the furrow advances at a rate of about 1 column ofclusters per 2 hr, it was estimated that dead cells fragment and arecleared within 2-4 hr.

[0120] The earliest defect observed in mutant discs is an increase inthe number of cells undergoing cell death before furrow formation. Noobvious morphological or structural abnormalities in progenitor cellsanterior to the furrow, other than changes characteristic of programmedcell death in dying cells, could be found by transmission electronmicroscopy. Furthermore, in alleles that make reduced eyes, progenitorcells that survive anterior to the furrow appear to enter into thenormal progression of events marked by the furrow. Together, theseresults suggest that the primary phenotype of loss of eye gene functionin the eye is the loss of progenitor cells through programmed cell deathprior to furrow formation.

Example 4 Transformation Rescue

[0121] To show definitively that the biological activity of the eya geneis encoded in the transcripts identified, transformation rescue of theeyeless phenotype was done. Type I cDNA was subcloned into the pHT4vector (Schneuwly et al., Nature 325:816-818 (1987)), downstream of theDrosophila hsp70 heat shock promoter, and injected into embryos, asoutlined below. Stable inserts were crossed into eya¹ and eya² mutantflies, which express viable eyeless phenotypes. Mutant larvae, harboringan hsp-eya minigene insert, were heat pulsed for 1 hr every 6-8 hr, fromthe first instar larval stage to pupation, to determine whetherexpression of the normal cDNA could restore eye development.

[0122] Type I cDNA was subcloned into the Kpnl site of the vector pHT4(Schneuwly et al., supra), to make an hsp-eya minigene fortransformation. The cDNA was first subcloned into the EcoRl site of amodified pBluescript vector with a Kpnl linker added to the Smal site(Van Vactor et al., supra). Partial digests were used, since the cDNAshave an internal Kpnl site. The pHT4 vector contains the Drosophilahsp70 promoter, a polyadenylation site from SV40, and ry⁺ gene as an eyecolor marker. The plasmid for transformation was cesium chloride-banded,then mixed at a 5:1 ratio with a transposase source, phsπ (Steller etal., EMBO J. 4:3765-3772 (1985)). This was injected into eggs (Rubin etal., Science 218:348-353 (1982)). Lines were established from adultsharboring an hsp-eya minigene insert, as assessed by eye color, thencrossed into eya mutant backgrounds. Two independent transformant lines,A23.4 and A67.1, were used for rescue of the eye phenotype. To expressthe cDNA during development, eggs were laid in vials, then after 24 hr,heat pulsed in a 37° C. water bath for 1 hr every 6-8 hr during larvaldevelopment, which was slowed to a period of about 7 days. For stagedlarvae, eggs were collected over 3 hr intervals, and larvae werecollected within 5 hr of the second or third instar larval molts. Larvaewere transferred to 0.5 ml tubes with cornmeal medium and were heatpulsed in a polymerase chain reaction machine for 30 min every 6 hr forthe desired number of pulses. For heat shocks during the pupal stage,white prepupae were collected every 1-2 hr over a 9 hr period from ry⁵⁰⁶and transformant lines A67.1 and B58.1 in a background. Pupae were aged15-24 hr, then heat shocked for 30 min every 6 hr for 48 hr. Althoughthis treatment caused much death of both control and experimentalanimals, eyes could be scored on emerged flies and with dissection ondead pupae. The period of heat shock covered the phase of pigment celldeath, which normally occurs 35-50 hr after pupation (Wolff et al.,supra).

[0123] The results show that mutant lines bearing the minigene andraised without heat shock were eyeless (FIG. 9B). Heat pulsing eya¹ andeya²mutant larvae lacking an insert had no effect on the mutantphenotype. However, heat pulsing of eya¹ and eya² mutant larvae with anhsp-eya minigene insertion restored the adult compound eye (FIG. 9C).Tangential sections through rescued eyes showed that the restoredommatidia appear normal (FIG. 9E). No discernible dominant effects ofheat shock-induced expression of the hsp-eya minigene were evident innormal flies or in eya mutants, other than rescue of the eye in thelatter.

[0124] The effective period of heat shock-induced rescue of the eyamutant phenotype was defined by heat pulsing during the second and thirdinstar larval stages. Heat pulses during only the second instar larvalstage were ineffective. However, eyes of small size could be restoredwith three 30 min heat pulses every 6 hr starting from the third instarlarval molt (data not shown), the stage during which the morphogeneticfurrow begins. With four or five heat pulses, eyes of intermediate sizecould be restored, and, additionally, were restored to a greaterpercentage of transformants. These data suggest that eya activity isrequired during the stage of development at which some cell deathnormally occurs anterior to the furrow.

[0125] Rescue of the eya mutant phenotype with the heat shock cDNAconstruct shows that the protein encoded by the type I cDNA can providethe biological activity required to rescue the progenitor cells fromdeath and restore the sequence of patterning events that generate aneye.

What is claimed is:
 1. A recombinant programmed cell death antagonistprotein.
 2. A recombinant programmed cell death antagonist proteinaccording to claim 1 which has the sequence shown in FIG.
 7. 3. Anisolated nucleic acid encoding a programmed cell death antagonistprotein.
 4. The nucleic acid of claim 3 comprising DNA having thesequence shown in FIG.
 7. 5. An anti-sense nucleic acid comprisingnucleic acid capable of hybridizing to all or part of the sequence shownin FIG.
 7. 6. An expression vector comprising transcriptional andtranslational regulatory DNA operably linked to DNA encoding aprogrammed cell death antagonist protein.
 7. A host cell transformedwith an expression vector comprising a nucleic acid encoding aprogrammed cell death antagonist protein.
 8. A method of producing aprogrammed cell death antagonist protein comprising: a) culturing a hostcell transformed with an expression vector comprising a nucleic acidencoding a programmed cell death antagonist protein; and b) causingexpression of said nucleic acid to produce a recombinant programmed celldeath antagonist protein.